The era of 3D and spatial genomics
Authors: Britta A.M. Bouwman, Nicola Crosetto, Magda Bienko
Journal: Trends in Genetics (2022)

For clarification across the field, we propose to use the term '3D genomics' to denote the study of the spatial positioning of chromatin in the nucleus – including chromatin contacts and associations with nuclear landmarks – whereas the term 'spatial genomics' is used to refer to the study of genome sequence and 3D structure variation between cells in their native tissue context.Combining in situ barcode sequencing and ex situ genome sequencing for spatially resolved genomics is an emerging approach that can be leveraged to address fundamental research questions on intercell genome variability and for cancer diagnostics, allowing high-resolution mapping of intratumor heterogeneity.Together, spatial genomics and spatial transcriptomics promise to revolutionize both biology and medicine by providing unprecedented insights into the spatial organization of tissues and how fundamental cellular processes are orchestrated in multicellular organisms. Over a decade ago the advent of high-throughput chromosome conformation capture (Hi-C) sparked a new era of 3D genomics. Since then the number of methods for mapping the 3D genome has flourished, enabling an ever-increasing understanding of how DNA is packaged in the nucleus and how the spatiotemporal organization of the genome orchestrates its vital functions. More recently, the next generation of spatial genomics technologies has begun to reveal how genome sequence and 3D genome organization vary between cells in their tissue context. We summarize how the toolkit for charting genome topology has evolved over the past decade and discuss how new technological developments are advancing the field of 3D and spatial genomics. Over a decade ago the advent of high-throughput chromosome conformation capture (Hi-C) sparked a new era of 3D genomics. Since then the number of methods for mapping the 3D genome has flourished, enabling an ever-increasing understanding of how DNA is packaged in the nucleus and how the spatiotemporal organization of the genome orchestrates its vital functions. More recently, the next generation of spatial genomics technologies has begun to reveal how genome sequence and 3D genome organization vary between cells in their tissue context. We summarize how the toolkit for charting genome topology has evolved over the past decade and discuss how new technological developments are advancing the field of 3D and spatial genomics. Unlike the term 'spatial transcriptomics' [1.Crosetto N. et al.Spatially resolved transcriptomics and beyond.Nat. Rev. Genet. 2015; 16: 57-66Crossref PubMed Scopus (275) Google Scholar], which is now widely accepted [2.Zhuang X. Spatially resolved single-cell genomics and transcriptomics by imaging.Nat. Methods. 2021; 18: 18-22Crossref PubMed Scopus (43) Google Scholar], the term 'spatial genomics' has emerged only recently and is less clearly defined. By analogy to spatial transcriptomics, the term 'spatial genomics' may be used to indicate the study of how the genome sequence varies across multiple regions of a healthy or diseased tissue or organ, for example using multiregion sequencing to study intratumor genetic heterogeneity [3.Gerlinger M. et al.Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.N. Engl. J. Med. 2012; 366: 883-892Crossref PubMed Scopus (5541) Google Scholar]. On the other hand, since the development of high-throughput chromosome conformation capture (Hi-C) in 2009 [4.Lieberman-Aiden E. et al.Comprehensive mapping of long range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4591) Google Scholar], many efforts in the spatial genomics field have concentrated on charting the 3D organization of the genome inside the cell nucleus [5.Cardozo Gizzi A.M. A shift in paradigms: spatial genomics approaches to reveal single-cell principles of genome organization.Front. Genet. 2021; 12780822Crossref PubMed Scopus (0) Google Scholar,6.McCord R.P. et al.Chromosome conformation capture and beyond: toward an integrative view of chromosome structure and function.Mol. Cell. 2020; 77: 688-708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar]. To avoid confusion, we propose to use the term '3D genomics' to denote the study of the spatial arrangement of the genetic material in the nucleus, and relative to nuclear landmarks (see Glossary) such as the lamina and prominent nuclear bodies, whereas the term 'spatial genomics' refers to the study of how the genome sequence or the spatial positioning of the genome in the nucleus varies from one cell to another (and between different cell types) at defined locations in a tissue or organ in a multicellular organism (Figure 1A,B , Key figure). We briefly revisit the key methodologies that are at the heart of 3D genomics – which enable 3D genome organization to be studied within cell populations and single cells – and then focus on emerging technologies that are igniting the field of spatial genomics – allowing the variation of (3D) genome organization to be studied across multicellular tissues. For more detailed side-by-side comparisons of the methods that have advanced the field of 3D genomics we refer to several excellent recent reviews [6.McCord R.P. et al.Chromosome conformation capture and beyond: toward an integrative view of chromosome structure and function.Mol. Cell. 2020; 77: 688-708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7.Jerkovic I. Cavalli G. Understanding 3D genome organization by multidisciplinary methods.Nat. Rev. Mol. Cell Biol. 2021; 22: 511-528Crossref PubMed Scopus (40) Google Scholar, 8.Kempfer R. Pombo A. Methods for mapping 3D chromosome architecture.Nat. Rev. Genet. 2020; 21: 207-226Crossref PubMed Scopus (165) Google Scholar, 9.Tjalsma S.J. de Laat W. Novel orthogonal methods to uncover the complexity and diversity of nuclear architecture.Curr. Opin. Genet. Dev. 2021; 67: 10-17Crossref PubMed Scopus (2) Google Scholar, 10.Sparks T.M. et al.Evolving methodologies and concepts in 4D nucleome research.Curr. Opin. Cell Biol. 2020; 64: 105-111Crossref PubMed Scopus (3) Google Scholar, 11.Jung N. Kim T.-K. Advances in higher-order chromatin architecture: the move towards 4D genome.BMB Rep. 2021; 54: 233-245Crossref PubMed Scopus (0) Google Scholar]. We also do not discuss multiregion sequencing approaches that have been widely deployed to study genome variation with spatial resolution in tumors. Most early studies on 3D genome organization used imaging-based approaches [12.Lamond A.I. Earnshaw W.C. Structure and function in the nucleus.Science. 1998; 280: 547-553Crossref PubMed Scopus (769) Google Scholar,13.Cremer T. Cremer C. Chromosome territories, nuclear architecture and gene regulation in mammalian cells.Nat. Rev. Genet. 2001; 2: 292-301Crossref PubMed Scopus (1659) Google Scholar], and the development of the first genomic method to query the 3D genome over two decades ago – chromosome conformation capture (3C) [14.Dekker J. et al.Capturing chromosome conformation.Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2433) Google Scholar] – created a massive ripple effect across the field. 3C and its follow-up high-throughput version, Hi-C [4.Lieberman-Aiden E. et al.Comprehensive mapping of long range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4591) Google Scholar], are two members of a large and expanding family of 3C-based methods [6.McCord R.P. et al.Chromosome conformation capture and beyond: toward an integrative view of chromosome structure and function.Mol. Cell. 2020; 77: 688-708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,7.Jerkovic I. Cavalli G. Understanding 3D genome organization by multidisciplinary methods.Nat. Rev. Mol. Cell Biol. 2021; 22: 511-528Crossref PubMed Scopus (40) Google Scholar,9.Tjalsma S.J. de Laat W. Novel orthogonal methods to uncover the complexity and diversity of nuclear architecture.Curr. Opin. Genet. Dev. 2021; 67: 10-17Crossref PubMed Scopus (2) Google Scholar] (Box 1) that all probe chromatin contacts between spatially proximal DNA sequences using proximity ligation. Application of these methods to a plethora of cell types and model organisms revealed that the genome is universally organized into various types of structural units, including A/B compartments and subcompartments [4.Lieberman-Aiden E. et al.Comprehensive mapping of long range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4591) Google Scholar,15.Rao S.S.P. et al.A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3326) Google Scholar], topologically associating domains (TADs) [16.Dixon J.R. et al.Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (3749) Google Scholar], and chromatin loops [15.Rao S.S.P. et al.A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3326) Google Scholar]. However, most 3C-based methods including Hi-C have several inherent biases owing to the use of proximity ligation, formaldehyde crosslinking, and restriction endonucleases [17.Chandradoss K.R. et al.Biased visibility in Hi-C datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide.BMC Genomics. 2020; 21: 175Crossref PubMed Scopus (4) Google Scholar, 18.Williamson I. et al.Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.Genes Dev. 2014; 28: 2778-2791Crossref PubMed Scopus (0) Google Scholar, 19.Gavrilov A.A. et al.Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub.Nucleic Acids Res. 2013; 41: 3563-3575Crossref PubMed Scopus (67) Google Scholar, 20.Yaffe E. Tanay A. Probabilistic modeling of Hi-C contact maps eliminates systematic biases to characterize global chromosomal architecture.Nat. 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To overcome some of these biases, various alternative techniques have been developed, including genome architecture mapping (GAM) [22.Beagrie R.A. et al.Complex multi-enhancer contacts captured by genome architecture mapping.Nature. 2017; 543: 519-524Crossref PubMed Scopus (323) Google Scholar], split-pool recognition of interactions by tag extension (SPRITE) [23.Quinodoz S.A. et al.Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus.Cell. 2018; 174: 744-757Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar], chromatin interaction analysis via droplet-based and barcode-linked sequencing (ChIA-Drop) [24.Zheng M. et al.Multiplex chromatin interactions with single-molecule precision.Nature. 2019; 566: 558-562Crossref PubMed Scopus (99) Google Scholar], DNA adenine methyltransferase (Dam)C [25.Redolfi J. et al.DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.Nat. Struct. Mol. Biol. 2019; 26: 471-480Crossref PubMed Scopus (0) Google Scholar], and chemical-crosslinking assisted proximity capture (CAP-C) [110.You Q. et al.Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution.Nat. Biotechnol. 2021; 39: 225-235Crossref PubMed Scopus (2) Google Scholar]. While GAM circumvents proximity ligation by inferring Hi-C-like proximity matrices from the frequency with which DNA loci are cosequenced in cryosections of crosslinked nuclei [22.Beagrie R.A. et al.Complex multi-enhancer contacts captured by genome architecture mapping.Nature. 2017; 543: 519-524Crossref PubMed Scopus (323) Google Scholar]. SPRITE partitions crosslinked chromatin complexes via successive rounds of split-and-pool barcoding, during which crosslinked chromatin fragments in the same complex 'travel' together and are tagged with the same serial barcode [23.Quinodoz S.A. et al.Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus.Cell. 2018; 174: 744-757Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. Similarly, ChIA-Drop relies on physically separating crosslinked and DNase I-digested chromatin complexes by droplet encapsulation using a microfluidic device [26.Ma S. et al.Microfluidics for genome-wide studies involving next generation sequencing.Biomicrofluidics. 2017; 11021501Crossref Scopus (19) Google Scholar]. This allows unique barcoding and amplification of co-crosslinked chromatin fragments, which potentially partake in meaningful contacts, followed by sequencing [24.Zheng M. et al.Multiplex chromatin interactions with single-molecule precision.Nature. 2019; 566: 558-562Crossref PubMed Scopus (99) Google Scholar]. By contrast, DamC represents the first crosslinking- and ligation-free alternative to 3C-based methods. DamC is a modification of Dam identification (DamID), an approach that involves fusing the Escherichia coli Dam methylase to a protein of interest, which upon expression leads to adenine methylation of GATC sequences that are spatially close to the fusion protein, enabling their identification by sequencing [27.van Steensel B. Henikoff S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase.Nat. Biotechnol. 2000; 18: 424-428Crossref PubMed Scopus (403) Google Scholar]. In DamC, Dam fused to reverse tetracycline receptors is conditionally recruited (upon treatment with doxycycline) to tetracycline operator arrays integrated into diverse genomic loci, after which sequencing of methylated DNA uncovers chromosomal contacts [25.Redolfi J. et al.DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.Nat. Struct. Mol. Biol. 2019; 26: 471-480Crossref PubMed Scopus (0) Google Scholar]. Because it is performed in non-fixed cells, DamC was used to demonstrate that TADs and specific loops are not an artifact of fixation and can be detected in vivo [25.Redolfi J. et al.DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.Nat. Struct. Mol. Biol. 2019; 26: 471-480Crossref PubMed Scopus (0) Google Scholar]. More recently, CAP-C [110.You Q. et al.Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution.Nat. Biotechnol. 2021; 39: 225-235Crossref PubMed Scopus (2) Google Scholar] set out to circumvent potential biases introduced by crosslinking of proteins to DNA (which occurs during formaldehyde crosslinking in most 3C-based methods). Such biases include the potentially hindering effects that DNA-bound proteins may have in masking restriction sites and obstructing proximity ligation, which in turn can reduce assay resolution and noise. To this end, CAP-C captures chromatin conformation with direct DNA–DNA crosslinking using a multifunctional chemical platform and UV irradiation to generate contact maps at sub-kilobase resolution and with low background noise [110.You Q. et al.Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution.Nat. Biotechnol. 2021; 39: 225-235Crossref PubMed Scopus (2) Google Scholar]. Moreover, when used without mild formaldehyde prefixing, CAP-C enables the detection of native chromatin conformations [110.You Q. et al.Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution.Nat. Biotechnol. 2021; 39: 225-235Crossref PubMed Scopus (2) Google Scholar]. Beyond pairwise contacts, GAM, SPRITE, ChIA-Drop, and several 3C methods [7.Jerkovic I. Cavalli G. Understanding 3D genome organization by multidisciplinary methods.Nat. Rev. Mol. Cell Biol. 2021; 22: 511-528Crossref PubMed Scopus (40) Google Scholar,9.Tjalsma S.J. de Laat W. Novel orthogonal methods to uncover the complexity and diversity of nuclear architecture.Curr. Opin. Genet. Dev. 2021; 67: 10-17Crossref PubMed Scopus (2) Google Scholar] also allow the detection of multiple contacts which may reveal higher-order chromatin structures, particularly when applied to single cells, as in single-cell SPRITE (scSPRITE) [28.Arrastia M.V. et al.Single-cell measurement of higher-order 3D genome organization with scSPRITE.Nat. Biotechnol. 2022; 40: 64-73Crossref PubMed Scopus (7) Google Scholar].Box 1A brief timeline of capturing chromatin conformationIn 2002, chromosome conformation capture (3C) [14.Dekker J. et al.Capturing chromosome conformation.Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2433) Google Scholar] was the first proximity ligation-based method for identifying the frequency of chromatin contacts across a cell population. 3C quantifies ligation events between pairs of restriction fragments harboring a priori selected genomic loci ('one versus one') using semi-quantitative PCR.In 2006, (circular) chromosome conformation capture (-on-chip) (4C) incorporated an inverse PCR step to amplify all chromatin fragments genome-wide that reside in spatial proximity to one (or several) selected genomic viewpoint fragment ('one versus all'), followed by analysis via microarrays [103.Simonis M. et al.Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C).Nat. Genet. 2006; 38: 1348-1354Crossref PubMed Scopus (978) Google Scholar] or sequencing [104.Zhao Z. et al.Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.Nat. Genet. 2006; 38: 1341-1347Crossref PubMed Scopus (684) Google Scholar]. Several 4C variants have been developed, including multi-contact 4C (MC-4C) [105.Vermeulen C. et al.Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies.Nat. Protoc. 2020; 15: 364-397Crossref PubMed Scopus (13) Google Scholar] for uncovering multi‐way contacts representing local cooperative and competitive chromatin topologies.In 2009, the first version of high-throughput chromosome conformation capture (Hi-C) [4.Lieberman-Aiden E. et al.Comprehensive mapping of long range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4591) Google Scholar] was devised for capturing chromatin contacts between loci genome-wide ('all versus all') in an unbiased manner. The initial megabase resolution increased significantly to kilobase resolution with the development of in situ Hi-C [15.Rao S.S.P. et al.A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3326) Google Scholar], Hi-C 2.0 [106.Belaghzal H. et al.Hi-C 2.0: an optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation.Methods. 2017; 123: 56-65Crossref PubMed Scopus (137) Google Scholar], and Hi-C 3.0 [107.Akgol Oksuz B. et al.Systematic evaluation of chromosome conformation capture assays.Nat. Methods. 2021; 18: 1046-1055Crossref PubMed Scopus (4) Google Scholar], whereas recent approaches such as Micro-C [108.Hsieh T.-H.S. et al.Mapping nucleosome resolution chromosome folding in yeast by Micro-C.Cell. 2015; 162: 108-119Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar] and Micro-C-XL [109.Krietenstein N. Rando O.J. Mammalian Micro-C-XL.Methods Mol. Biol. 2022; 2458: 321-332Crossref PubMed Scopus (0) Google Scholar] use micrococcal nuclease instead of restriction endonucleases to enable 3D genomics at nucleosome resolution.In 2013, the first single-cell Hi-C (scHi-C) [47.Nagano T. et al.Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.Nature. 2013; 502: 59-64Crossref PubMed Scopus (906) Google Scholar] protocol became available to generate genome-wide chromatin contact maps of single cells. To reduce loss of material and increase resolution, more recent approaches such as Dip-C [49.Tan L. et al.Three-dimensional genome structures of single diploid human cells.Science. 2018; 361: 924-928Crossref PubMed Scopus (185) Google Scholar] incorporate whole-genome amplification on single nuclei combined with transposon-based library preparation. Dip-C represents the most powerful scHi-C available to date, and has 20 kb or 100 nm resolution.In 2019, a crosslinking and ligation-free alternative to 4C, DamC [25.Redolfi J. et al.DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.Nat. Struct. Mol. Biol. 2019; 26: 471-480Crossref PubMed Scopus (0) Google Scholar], was established. DamC builds on the DamID technique [27.van Steensel B. Henikoff S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase.Nat. Biotechnol. 2000; 18: 424-428Crossref PubMed Scopus (403) Google Scholar,32.Guelen L. et al.Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1287) Google Scholar] and uses ectopic viewpoints to which Dam is recruited to methylate nearby genomic regions, thereby earmarking them to be detected by sequencing.In 2021, chemical-crosslinking assisted proximity capture (CAP-C) [110.You Q. et al.Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution.Nat. Biotechnol. 2021; 39: 225-235Crossref PubMed Scopus (2) Google Scholar] was devised for capturing chromatin conformation with a direct DNA–DNA crosslinking approach using a multifunctional chemical platform consisting of poly(amidoamine) (PAMAM) dendrimers, psoralen, and UV irradiation. Compared to 3C approaches that use formaldehyde, CAP-C generates chromatin contact maps at sub-kilobase resolution with low background noise.For a comprehensive overview of the possibilities and limitations of these and other 3C-derived methods and ligation-free alternatives, and summaries of how they have contributed to enrich our knowledge of the 3D genome, we refer to excellent recent reviews [7.Jerkovic I. Cavalli G. Understanding 3D genome organization by multidisciplinary methods.Nat. Rev. Mol. Cell Biol. 2021; 22: 511-528Crossref PubMed Scopus (40) Google Scholar,9.Tjalsma S.J. de Laat W. Novel orthogonal methods to uncover the complexity and diversity of nuclear architecture.Curr. Opin. Genet. Dev. 2021; 67: 10-17Crossref PubMed Scopus (2) Google Scholar, 10.Sparks T.M. et al.Evolving methodologies and concepts in 4D nucleome research.Curr. Opin. Cell Biol. 2020; 64: 105-111Crossref PubMed Scopus (3) Google Scholar, 11.Jung N. Kim T.-K. Advances in higher-order chromatin architecture: the move towards 4D genome.BMB Rep. 2021; 54: 233-245Crossref PubMed Scopus (0) Google Scholar]. In 2002, chromosome conformation capture (3C) [14.Dekker J. et al.Capturing chromosome conformation.Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2433) Google Scholar] was the first proximity ligation-based method for identifying the frequency of chromatin contacts across a cell population. 3C quantifies ligation events between pairs of restriction fragments harboring a priori selected genomic loci ('one versus one') using semi-quantitative PCR. In 2006, (circular) chromosome conformation capture (-on-chip) (4C) incorporated an inverse PCR step to amplify all chromatin fragments genome-wide that reside in spatial proximity to one (or several) selected genomic viewpoint fragment ('one versus all'), followed by analysis via microarrays [103.Simonis M. et al.Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C).Nat. Genet. 2006; 38: 1348-1354Crossref PubMed Scopus (978) Google Scholar] or sequencing [104.Zhao Z. et al.Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.Nat. Genet. 2006; 38: 1341-1347Crossref PubMed Scopus (684) Google Scholar]. Several 4C variants have been developed, including multi-contact 4C (MC-4C) [105.Vermeulen C. et al.Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies.Nat. Protoc. 2020; 15: 364-397Crossref PubMed Scopus (13) Google Scholar] for uncovering multi‐way contacts representing local cooperative and competitive chromatin topologies. In 2009, the first version of high-throughput chromosome conformation capture (Hi-C) [4.Lieberman-Aiden E. et al.Comprehensive mapping of long range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4591) Google Scholar] was devised for capturing chromatin contacts between loci genome-wide ('all versus all') in an unbiased manner. The initial megabase resolution increased significantly to kilobase resolution with the development of in situ Hi-C [15.Rao S.S.P. et al.A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3326) Google Scholar], Hi-C 2.0 [106.Belaghzal H. et al.Hi-C 2.0: an optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation.Methods. 2017; 123: 56-65Crossref PubMed Scopus (137) Google Scholar], and Hi-C 3.0 [107.Akgol Oksuz B. et al.Systematic evaluation of chromosome conformation capture assays.Nat. Methods. 2021; 18: 1046-1055Crossref PubMed Scopus (4) Google Scholar], whereas recent approaches such as Micro-C [108.Hsieh T.-H.S. et al.Mapping nucleosome resolution chromosome folding in yeast by Micro-C.Cell. 2015; 162: 108-119Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar] and Micro-C-XL [109.Krietenstein N. Rando O.J. Mammalian Micro-C-XL.Methods Mol. Biol. 2022; 2458: 321-332Crossref PubMed Scopus (0) Google Scholar] use micrococcal nuclease instead of restriction endonucleases to enable 3D genomics at nucleosome resolution. In 2013, the first single-cell Hi-C (scHi-C) [47.Nagano T. et al.Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.Nature. 2013; 502: 59-64Crossref PubMed Scopus (906) Google Scholar] protocol became available to generate genome-wide chromatin contact maps of single cells. To reduce loss of material and increase resolution, more recent approaches such as Dip-C [49.Tan L. et al.Three-dimensional genome structures of single diploid human cells.Science. 2018; 361: 924-928Crossref PubMed Scopus (185) Google Scholar] incorporate whole-genome amplification on single nuclei combined with transposon-based library preparation. Dip-C represents the most powerful scHi-C available to date, and has 20 kb or 100 nm resolution. In 2019, a crosslinking and ligation-free alternative to 4C, DamC [25.Redolfi J. et al.DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.Nat. Struct. Mol. Biol. 2019; 26: 471-480Crossref PubMed Scopus (0) Google Scholar], was established. DamC builds on the DamID technique [27.van Steensel B. Henikoff S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase.Nat. Biotechnol. 2000; 18: 424-428Crossref PubMed Scopus (403) Google Scholar,32.Guelen L. et al.Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1287) Google Scholar] and uses ectopic viewpoints to which Dam is recruited to methylate nearby genomic regions, thereby earmarking them to be detected by sequencing. In 2021, chemical-crosslinking assisted proximity capture (CAP-C) [110.You Q. et al.Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution.Nat. Biotechnol. 2021; 39: 225-235Crossref PubMed Scopus (2) Google Scholar] was devised for capturing chromatin conformation with a direct DNA–DNA crosslinking approach using a multifunctional chemical platform consisting of poly(amidoamine) (PAMAM) dendrimers, psoralen, and UV irradiation. Compared to 3C approaches that use formaldehyde, CAP-C generates chromatin contact maps at sub-kilobase resolution with low background noise. For a comprehensive overview of the possibilities and limitations of these and other 3C-derived methods and ligation-free alternatives, and summaries of how they have contributed to enrich our knowledge of the 3D genome, we refer to excellent recent reviews [7.Jerkovic I. Cavalli G. Understanding 3D genome organization by multidisciplinary methods.Nat. Rev. Mol. Cell Biol. 2021; 22: 511-528Crossref PubMed Scopus (40) Google Scholar,9.Tjalsma S.J. de Laat W. Novel orthogonal methods to uncover the complexity and diversity of nuclear architecture.Curr. Opin. Genet. Dev. 2021; 67: 10-17Crossref PubMed Scopus (2) Google Scholar, 10.Sparks T.M. et al.Evolving methodologies and concepts in 4D nucleome research.Curr. Opin. Cell Biol. 2020; 64: 105-111Crossref PubMed Scopus (3) Google Scholar, 11.Jung N. Kim T.-K. Advances in higher-order chromatin architecture: the move towards 4D genome.BMB Rep. 2021; 54: 233-245Crossref PubMed Scopus (0) Google Scholar]. In addition to methods that sequence DNA–DNA contacts, several other assays chart genome organization in relation to nuclear landmarks (Box 2). For example, RNA and DNA (RD)-SPRITE allows the simultaneous capture of RNA–DNA, RNA–RNA, and DNA–DNA contacts, thus enabling the detection of chromatin hubs associated with nuclear b